Like all herpersviruses, herpes simplex virus-1 (HSV-1) consists of an icosahedral capsid surrounded by a membrane envelope. The capsid contains the viral DNA while a layer of protein called the tegument is found between the capsid and the membrane. Morphogenesis of the virus begins with assembly of the capsid which takes place in the infected cell nucles. A DNA-free capsid shell is first formed and them packaged with the viral dsDNA. Capsid assembly involves condensation of a major acpsid protein (VP5), three other structural proteins (VP19C, VP23 and Vp26), the scaffolding protein (preVP22A), the protease and several proteins present in smaller amounts. The proposed project is designed to clarify the sequence of zsteps required for capsid formation by making use of a cell-free capsid assembly system. The project is undertaken with the idea that idividual steps in capsid assembly are appropriate targetsfor the design of novel therapeutics since formation of the capsid is required for HSV-1 replication. In its basic features, the pathway of capsid assembly in HSV-1 is expeccted to be the same as that for other herpes viruses. Thus, potential drug targets identified in HSV-1 should be able to be exploited for the design of therapeutics effective against other herpes viruses including human cytomegalovirus, varicella-zoster virus, Epstein-Barr virus and Kaposi's sarcoma herpes virus. The specific goals of the project are to: (1) create an in vitro system in which HSV-1 capsids can be formed from purified proteins; (2) use cryoelectron microscopy and three-dimensional image reconstruction to determine the structures of morphological intermediates in the transitionbetween the procapsid and the mature polyhedral capsid; (3) examine HSV-1-infected cells for the presence of the procapsid, the spherical intermediate identified during cell-free capsid formation; (4) identify specific regions of VP19C required for capsid formation; and (5) use cryoelectron microscopy and three- dimensional image reconstruction to study attachment of tegument proteins to the capsid as it occurs in tegument-containing capsids isolated after treatment of native virus with Triton X-100.